Growth of hybridomas (immortalized antibody secreting cells) and lymphoid cells (immortal cells often used as fusion partners in constructing hybridomas) in serum-free media is very desirable. Serum is difficult and expensive to obtain, store and use; it is a source of undesirable foreign proteins which may be carried into the final product; its composition varies from lot to lot; and it is a potential source of contaminating organisms.
The present invention is concerned with culturing cells, in clouding mammalian cells, using a chemically-defined medium. The term "chemically-defined medium" is used in tissue culture to refer to culture media of known chemical composition, both quantitatively and qualitatively, in contrast to those media which contain natural products such as animal serum, embryo extracts, yeast hydrolysates, lactalbumin hydrolysates, tryptose or tryptone. In its strictest definition, it excludes high molecular weight proteins such as albumin which tightly bind other proteins and lipids which may resist purification.
A number of chemically-defined media are known. Most of these are solutions of carbohydrates, lipids, amino acids, vitamins, salts, minerals, purine and pyrimidine bases, etc. Some of these are widely used, for example, Dulbecco's modified Eagle's medium, In Vitro 6:89 (1970); Virology 8:396 (1959); Virology 12:185 (1960); Ham's F12 medium, Proc. Natl. Acad Sci. U.S.A. 63:288 (1965); In Vitro 6:89 (1970). Often one or more of these "basal media" are mixed in various proportions (e.g. Dulbecco's modified Eagle's medium: Ham's F12 medium, 1:1, vol:vol) before use, to obtain optimal growth for a given cell line.
Most basal media by themselves are ineffective at supporting the growth of most mammalian cells. The addition of supplemental factors, including transferrin, hydrocortisone, insulin, epidermal growth factor, ethanolamine, selenium and others is routinely required to obtain continuous growth of most mammalian cells in defined medium (Barnes and Sato, Anal. Biochem. 102:255 (1980)). Often the origin of the cell line under study dictates which supplements must be included to obtain optimal growth.
The formulation of defined media for the growth of lymphoid cells is commercially important for many reasons. Most established lymphoid cell lines secrete proteins, including interferons, interleukins, and antibodies, which have commercial applications. When the formulation of a defined medium for the growth of mammalian lymphoid cells was attempted, it was thought that serum albumin and 2-mercaptoethanol were necessary constituents in such preparations. More recently, it was discovered that catalase and dilinoleoyl phosphatidylcholine could replace albumin and 2-mercaptoethanol. Dilinoleoyl phosphotidylcholine supplied the requirement for an unsaturated fatty acid; catalase degraded harmful hydrogen peroxide that was present in basal tissue culture media. Also, it was learned that transferrin, selenium and ethanolamine were important for the growth of lymphoid cells in defined medium (Darfler and Insel, J. Cell. Physiol 115:31 (1983), Murakami et al, Anal. Biochem. 114:422 (1981), Iscove et al, Exp. Cell Res. 126:121 (1980), Chang et al, J. Immunol. Meth., 39:369 (1980)). Despite the progress in the development of defined media for lymphoid cells, many such preparations were unsatisfactory in that they displayed significant "dilution death", a term used to describe cell death resulting from diluting a culture of cells at high cell density (e.g. 1.times.10.sup.6 cells/ml) to one of low cell density (less than 1.times.10.sup.3 cells/ml).
As reported by McHugh, et al., BioTechniques, 72,76 (June/July 1983), hybridoma cells do not survive or grow well when plated at very low densities in serum free medium. Growth is particularly a problem in low density suspension cultures in serum-free media. Cleveland, et al., J. Immunol. Meth., 56:221 (1983).
While the inclusion of catalase in the medium offered some protection against dilution death, it was incompletely effective for certain cell lines using certain basal media; cloning efficiencies were 20% of maximum. Zigler et al (In Vitro Cell. and Dev. Biol. 21:282 (1985)) have shown that catalase was incompletely effective in protecting against toxicity due to media exposed to ordinary room light, suggesting the formation of toxic compounds that are not degraded by catalase. Similar results were reported by Wang and Nixon (In Vitro 14:715 (1978)).
Other groups (e.g. Kawamoto et al., Anal. Biochem. 130:445 (1985)) have attempted to overcome these problems by the inclusion of sulfhydryl-containing compounds such as 2-mercaptoethanol complexed to albumin; these two compounds together supplied reducing power to the medium. Similarly, cysteine has been used to supply the thiol requirement for serum-containing media (Broome and Jeng, J. Exp. Med. 138:574 (1973)). Cysteine, however, is less effective than 2-mercaptoethanol, probably owing to its more rapid degradation in tissue culture media.
Intensive efforts have been expended to discover formulations of serum-free media that are chemically-defined, stable, able to continuously grow lymphoid cells at clonal and high cell densities and which contain little or no high molecular weight proteins such as albumin.
Cartaya, U.S. Pat. No. Re. 30,985; Stemerman, U.S. Pat. No. 4,443,546 Torney, U.S. Pat. No. 3,887,403; and Weiss, U.S. Pat. No. 4,072,565 relate generally to serum-free media, which may contain the amino acids cysteine or cystine. Chang, et al., J. Immunol. Meth., 39:364-75 (1980) describe the growth of hybridomas in serum-free media. Murakami, et al., PNAS, 79:1158 (1982) report that ethanolamine was a necessary component of serum-free hybridoma growth media.
Darfler, et al., PNAS, 77:5993 (1980) described a new serum-free medium, containing casein, insulin, transferrin, testosterone, and linoleic acid, for growing murine lymphomas. They reported that 2-mercaptoethanol did not enhance growth in this "CITTL" medium. They later showed that CITTL was a medium suitable for the cultivation of a wide variety of transformed lymphoid cells, including hybridomas. Darfler and Insel, Exper. Cell Res., 138:287 (1982); Darfler and Insel, in Sato, ed., GROWTH OF CELLS IN NORMALLY DEFINED MEDIA, at 717 (1982).
Darfler and Insel, J. Cellular Physiol., 115:31 (1983) found that clonal growth of murine S49 T lymphoma cell lines required elimination of H.sub.2 O.sub.2 toxicity, and used catalase as a scavenger for H.sub.2 O.sub.2 in their serum-free medium.
Darfler, "In Vitro Immunization for the Generation of Hybridomas Using Serum-Free Medium", appearing in Bartal, ed., HYBRIDOMA FORMATION: MECHANISMS AND TECHNICAL ASPECTS OF HYBRIDOMA GENERATION AND MONOCLONAL ANTIBODY PRODUCTION (Humana Press, 1985) similarly overcame the problem of hybridoma susceptibility of hydrogen peroxide-mediated cytotoxicity. The growth medium included cysteine, but there was no teaching of any relationship of cysteine to inhibition of cytotoxicity. See also Darfler and Insel, in METHODS FOR SERUM-FREE CULTURE OF NEURAL AND LYMPHOID CELLS, 187 (1984).
Darfler and Insel showed that catalase can be used in serum-free tissue culture media for lymphoid cell growth to break down hydrogen peroxide to water and oxygen and prevent some of the damage of oxidizing, toxic agents. Catalase is less than satisfactory because it is a large (360,000 daltons) protein and, like albumin, its inclusion in tissue culture media hinders purification of proteins (e.g. antibodies) that are secreted by cultured lymphoid cells. Also catalase is incompletely effective at preventing damage to lymphoid cells in serum-free media when those cells are diluted to very low cell densities. Finally, catalase is potentially immunogenic and is unsuitable for use in tissue culture media intended for in vivo human uses and is less desirable for in vitro immunization protocols where levels of antigen may be in ng/ml levels.
Glutathione is known to play an important role in protecting cells against the destructive effects of reactive oxygen intermediates (such as H.sub.2 O.sub.2) and free radicals. Tsan, et al., Biochem. & Brophys. Res. Commun., 127:270 (Feb. 28, 1985). Since cysteine is used for glutathione synthesis, inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine depletes intracellular gluthathione levels and may result in cell damage due to oxidants. Contrariwise, an intracellular cysteine delivery system may be used to promote glutathione synthesis and thus protect the cell. Williamson, PNAS, 79:6246 (1982); Wellner, PNAS 81:4732 (1984). Thor, et al., Arch. Biochem. Brophys., 192:405 (1979) reported that cysteine, N-acetylycysteine and methionine protect hepatocytes from bromobenzene toxicity by providing intracellular cysteine for gluthathione biosynthesis.
Wellner et al. showed that the inclusion of 5 mM reduced glutathione ester to a medium composed of RPMI 1640 plus fetal calf serum raised intracellular glutathione levels for up to 7 days and concomitantly protected human lymphoid cells from damage due to radiation. The effort to find such compounds that raise intracellular glutathione levels was for the purpose of using them for the detoxification of the liver and kidney. The glutathione ester described was readily susceptible to oxidation and hydrolysis and "appreciable oxidation" of the molecule occurs as well as "some cleavage of the ester group" under ordinary culture conditions. In a related study, Tsan et al showed that L-2-oxothiazolidine-4-carboxylate (a precursor of cysteine) can raise intracellular cysteine (and, hence, glutathione) levels in pulmonary artery endothelial cells. Levels of intracellular glutathione were less than 170% of control; when the cells were treated with hydrogen peroxide, only a slight protective effect on cell death resulted. L-2-oxothiazolidine-4-carboxylate contains no free sulfhydryl groups and, as a result, would not be expected to have any protective effects extracellularly against oxidizing, toxic agents. The use of L-2-oxothiazolidine-4-carboxylate as an inclusion to tissue culture medium has been suggested as a means to obviate the toxicity of cysteine in certain cells (Williamson et al., PNAS 79:6246 (1982)).
Taylor, "Toxicity and Hazards to Successful Culture: Cellular Responses To Damage Induced By Light, Oxygen Or Heavy Metals", appearing in Patterson, ed., USES AND STANDARDIZATION OF VERTEBRATE CELL CULTURE RESEARCH (Tissue Culture Association Monograph, No. 5, 1984), has reviewed the toxicity and hazards of cell culture, focusing on damage from light, oxygen and heavy metals. He summarizes those endogenous (in vivo) mechanisms which mitigate oxygen toxicity, including those mediated by pH, ceruloplasmin, transferrin, trace metals, superoxide dismutase, catalase, glutathione, glutathione peroxidase, vitamin E, cysteine and ascorbic acid. He proposed the "use of `antioxidants`, compounds that either terminate the chain of oxidative reactions or combine with free radicals to absorb and dissipate their energy nondestructively (quenchers, scavengers, et cetera)." Specifically, for in vitro cell culture, he suggests the use of 2-mercaptoethanol, butylated hydroxytoluene, butylated hydroxyanisole, ascorbic acid, sodium selenite, dimethlysulfoxide, dimethylurea and vitamin E.
Hoffeld reported that 2-mercaptoethanol enhances the availability of glutathione, and that glutathione then directly scavenges radicals and peroxides intracellularly. Eur. J. Immunol., 11:371 (1981); See also Hoffeld and Oppenheim, Eur. J. Immonol., 10:391 (1980).
Bernard et al., J. Clin. Invest., 73:1772 (1984) reported a study on the use of N-acetylcysteine in the treatment of adult respiratory distress syndrome. They hypothesized that oxygen free radicals are released in this disease and that intravenous N-acetylcysteine was effective at relieving the symptoms of these toxic radicals using an animal model system. In vitro studies were performed to show that N-acetylcysteine, in a dose-dependent manner, with optimal inhibition observed at 17 mM, inhibited the chemiluminescence generated by either phorbol ester-stimulated leukocytes or a cell-free hydrogen peroxide-generating enzyme system containing human plasma and albumin. They hypothesized that N-acetylcysteine is a "direct free radical scavenger." No suggestion was made of the use of N-acetylcysteine as an additive to chemically defined serum-free tissue culture media to prevent the toxicity of endogenously-generated oxidizing agents. Bernard taught use of NAC at a level (17 mM) over an order of magnitude higher than the level of NAC found to be toxic to lymphoid cells grown in vitro (1-2 mM) and found the levels taught herein (0.1-1.0 mM) to be only marginally effective for his application.
Ormstad and Ohno, Cancer Res., 44:3797 (1984) reported that increasing the urinary excretion of compounds containing free thiol groups, particularly N-acetylcysteine and sodium 2-mercaptoethane sulfonate (MESNA), protects against cyclophosphamide toxicity. Cyclophosphamide is used as a cytostatic agent in cancer chemotherapy and as an immunosuppressant in organ transplantation. MESNA was the preferred protective agent.
Broome and Jeng, J. Exper. Med., 138:574 (1973) reported that a number of thiols and disulfides may be substituted for L-cysteine in serum-containing media for L1210 murine lymphoid cells. Among others, 3-mercaptopropionate, 2-mercaptoethanol and dithiothreitol were found to be effective, whereas DL-p enicillamine was ineffective. In addition, L-cysteine was active whereas D-cysteine was not. Other lymphoma lines were found to respond differently to thiols-disulfides in vitro.
Kendall & Hutchins and Goodman & Weigle studied the effects of D-penicillamine (D-PEN) on the [3H]-thymidine incorporation of mouse splenic lymphocytes in serum-containing (both groups) and serum-free (Goodman and Weigle) media. Optimal levels of D-PEN were 3.35 mM (Kendall & Hutchins) and 1-8 mM (Goodman & Weigle). In serum-free medium, the optimal level was 1-3 mM. Both the oxidized and reduced forms of D-PEN were mitogenic when used alone, but only the reduced form was active in the presence of another mitogen such as lipopolysaccharide (Goodman and Weigle). Dithiothreitol was also active (Kendall and Hutchins). Kendall and Hutchins showed data to support their hypothesis that D-PEN improved the culture medium . . . "by assisting reduction of L-cystine to L-cysteine, which is taken up by some cells more readily than the oxidized form [cystine]. . ." See Kendall & Hutchins, Immunol., 35:189 (1978); Goodman & Weigle, Cell Immunol. 65: 337 (1981).
The cell concentrations used in the above studies were on the order of 10.sup.6 cells/ml. The toxic effects of oxidants are not usually evident until cell concentrations are reduced to 10.sup.4 cells/ml. The growth promoting substances of the present invention do not act by increasing cysteine uptake but rather by inactivating oxidizing agents. Kendall and Hutchins teach that dithiothreitol is effective, while I have found it ineffective at the lower cell densities contemplated by the present invention.
Claesson, et al., Med. Microbiol. Immunol., 167:161 (1979) found that D-Penicillamine had an enhancing effect on .sup.3 H-TdR uptake by human spleen cells in serum-containing media at concentrations of 10.sup.-3 to 10.sup.-2 moles. Human spleen cells did not survive serum-free conditions.
Immortal or immortalized cells are already "mitogen activated" and the mitogenic effect observed with D-Penicillamine is apparent only when it is administered to normal lymphocytes.
Saville, Analyst, 83:670 (1958) describes a scheme for the colorimetric determination of microgram amounts of thiols over 0.02 mM. This method can be used even in the presence of large amounts of amino acids and, as such, is suitable for assaying thiol levels in tissue culture media.